Myocardial cell death induced by ischemia-reperfusion (IR) injury is a major cause of morbidity and mortality in the US. Mounting evidence suggests that the opening of a nonspecific inner mitochondrial membrane conductance channel, the mitochondrial Permeability Transition Pore (mPTP), is a key factor of IR injury. One effect of mPTP opening is the leakage of protons across the inner mitochondrial membrane, which uncouples mitochondrial oxidation from ATP synthesis and exacerbates the preexisting ATP deficit. Pharmacological inhibition of mPTP is a promising strategy for cardio protection in acute ischemic events. However, the lack of effective noninvasive methods to study mitochondrial function in vivo limits the understanding of IR injury mechanisms, as well as the development of novel therapeutics. Phosphorus-31 (31P) Magnetic Resonance Spectroscopy (MRS) is an attractive method to address this need. 31P Magnetization Transfer spectroscopy (MT-MRS) has long been proposed as a means of measuring ATP synthesis rates in vivo. Combination of 31P MT-MRS with the measurement of oxygen consumption rates allows the assessment of mitochondrial coupling. However, current 31P MT-MRS methods require prohibitively long imaging time to accurately measure ATP synthesis rates. On the other hand, recent development of Magnetic Resonance Fingerprinting (MRF) method provides a completely new framework for Magnetic Resonance data acquisition that allows simultaneous measurement of several tissue properties, including relaxation times, at high speed. The measurement of chemical exchange flux in MT-MRS is highly analogous to the measurement of relaxation times in MR imaging. Therefore, the overall objective of this proposal is to develop and validate fast novel 31P Magnetic Resonance Spectroscopy Fingerprinting (MRSF) technique to quantitatively measure ATP synthesis rate. This project has two specific aims. Aim 1 has three parts: developing 31P MRSF methods by computer simulation, implementing the developed methods in phantom experiments, and validating 31P MRSF methods against established 31P MT-MRS methods in perfused hearts under varying workloads. In Aim 2, the utility of the developed 31P MRSF method will be further assessed in a rat model of global IR injury, with and without pharmacological inhibition of mPTP. The results of these proposed experiments will help elucidate the mechanisms of mPTP inhibition induced cardio protection in IR. Furthermore, successful method development will pave the way for clinically feasible and relevant diagnostics as well as longitudinal studies of a spectrum of metabolic disorders.